The complexity of biological systems stems to a large extent from the high degree of interactions amongst their constituent components. As such, the cell is often described as a complex circuit consisting of an interacting network of molecules. Fusion proteins that function as molecular switches and serve to couple cellular functions are key components of this network. A switch recognizes an input signal (e.g. ligand concentration, pH, covalent modification) and, as a result, its output signal (e.g. enzyme activity, ligand affinity, oligomeric state) is modified. Examples of natural switches include allosteric enzymes which couple effector levels to enzymatic activity and ligand-dependent transcription factors that couple ligand concentration to gene expression. The ability to create novel switches or to modify existing switches by coupling previously uncoupled protein functions would enable the creation of selective protein therapeutics that are able to “sense” the cellular state and carryout the desired function conditionally depending on that state. In addition, the ability to create protein switches has tremendous practical potential for developing novel molecular sensors, medical diagnostics and as a tool for elucidating molecular and cellular functions. Additionally, such switches are an addition to the synthetic biologist's toolbox for creating programmable cells for biotechnological and bioengineering applications because they directly link the protein's specific activity to the cellular state.
There is recognition that there is great potential to design fusion proteins that act as molecular switches to modulate or report on biological functions for a variety of applications including biosensors, modulators of gene transcription and cell signaling pathways, and novel biomaterials. Despite its great potential, however, molecular switch technology has not been extensively exploited, in part due to technical challenges in engineering effective molecular switches. Most existing strategies for engineering switches involve the reprogramming of existing switches, the engineering of control over protein interactions the alleviating of the effects of deleterious mutations by the binding of small molecules, or the modulation of protein folding. In general, existing approaches to creating protein molecular switches include: control of oligomerization or proximity using chemical inducers of dimerization (CID); chemical rescue; fusion of the target protein to a steroid binding domain (SBD); coupling of proteins to non-biological materials or metal nanocrystals, and domain insertion.
Gene-directed enzyme prodrug therapy (GDEPT; also known as “suicide gene therapy”) is an emerging gene therapy strategy against cancer. In GDEPT, the gene encoding an enzyme, which can activate the prodrug, is delivered to cancer cells. This step is followed by the systemic administration of a prodrug. This prodrug is converted to the toxic drug by the enzyme. To the extent that the enzyme is produced only in cancer cells, the toxic drug will be produced only in cancer cells.
Current approaches to GDEPT attempt to achieve specificity in two ways. The specificity of the prodrug activation has to rely on either targeted delivery of the gene to the desired cancer cells (transductional targeting) or the ability to limit gene expression to the targeted cells (transcriptional targeting). Transductional targeting suffers from the difficulty in creating gene delivery vehicles that are both efficient and specific (since, in general, efficiency is sacrificed for specificity). Although systemic virus administration is likely to be more effective, all clinical GDEPT studies to date have utilized local administration of the viral vectors at or near the tumor site because of transductional targeting limitations. The extent to which normal tissues are transduced with the suicide gene limits the dose of prodrug that can be administers and the effectiveness of the treatment. Transcriptional targeting is a more recent approach that attempts to circumvent this problem either by using tumor-selective promoters to drive expression of one or more viral genes that regulate viral replication (hence tumor cells will have more copies of the suicide gene and thus produced more the prodrug-converting enzyme) or by using tumor-selective promoters to drive expression of the suicide gene. However, viral replication increases the risk of insertional mutagenesis and oncogenesis. The majority of successful studies using tumor-selective promoters have been preclinical animal model studies with uncertain relevance to human cancer. The success of this approach will depend on the promoter strength in tumor cells and the lack of transcription in normal cells. It is not clear at present whether the difference between the two will be sufficient for effective selectivity.
There remains a need in the art for better cancer therapeutics, and in particular, better methods to more efficiently treat cancer cells while reducing the side-effects associated with these treatments.